Transport of substances between cell organelles, which are unit membrane-enclosed structures such as endoplasmic reticulum, is conducted by intracellular vesicle transport. In endocrine cells including pancreatic β-cells and pituitary cells, peptides/proteins synthesized at ribosomes are received by the endoplasmic reticulum, from which they are transported in vesicles, which are transformed into secretory vesicles through the Golgi body and transported to the cell membrane, where they are released out of the cell via a step which includes fusion of the membranes. In neurons, neurotransmitter-containing precursors of synaptic vesicles are formed in Golgi bodies and transported by microtubules along the axon and stored at the synapse. Depolarization of the pre-synaptic membrane causes the vesicles to fuse with the pre-synaptic membrane and thus the neurotransmitters are released. This type of secretion based on the fusion of the vesicles and the cell membrane is called exocytosis.
In contrast, when extracellular substances such as hormones including cell growth factors are bound to the cell membrane, the complexes thus formed are invaginated into the cell to form endosomes. This type of uptake of environmental substances is called endocytosis.
Formation of vesicles, such as by budding, commonly observed both in exocytosis and endocytosis, and docking and fusion, the phenomena observed in process of their transportation and binding to other membrane systems, are regulated by a GTP-binding, low-molecular protein, called G protein. More than 30 types of this protein are known. The group of the proteins, which are also classified in Rab family, regulate the intracellular vesicle transport system.
With regard to the intracellular vesicle transport system, it is understood today that a cell is in a resting state when Rab protein occurs in a bound form to guanine nucleotide diphosphate (GDP), and that budding, docking and fusion are triggered as a result of a process in which a protein having GEF activity act on Rab protein and converts it to GTP-binding Rab protein, to which GTP binds to form a GTP-Rab complex, which in turn binds to a corresponding target protein on the membrane.
Stimulus-secretion coupling plays an important role in exocytosis observed in many cell types including neurons and endocrine cells [J. E. Rothman, Nature 372:55(1994); T. C. Sudhof, Nature 375:645 (1995)]. While a rise in intracellular Ca2+ concentration is important in the regulation of exocytosis, other signals are also known to play important roles. cAMP (cyclic adenosine-3′,5′-monophosphate)/PKA (cAMP-dependent protein kinase A) signaling pathway is known to regulate exocytosis in many of neurons, neuroendocrine cells and endocrine cells. In particular, cAMP has been thought to mediate long-term potentiation by increasing neurotransmitter release in the brain [R. D. Hawkins et al. Ann. Rev. Neurosci. 16:625(1993); G. Lonart et al., Neuron 21:1141(1998)]. cAMP also regulates exocytosis responsible for insulin release from pancreatic β-cells and amylase release from parotid acinar cells [P. M. Jones and S. J., Persaud, Endocrine. Rev. 19:429(1998); E. Renstrom, et al., J. Physiol. 502:105(1997); K. Yoshimura, Biochim. Biophys. Acta 1402:171(1998)].
In addition to its role in PKA-dependent phosphorylation of regulatory proteins associated with the process of exocytosis, it is known that cAMP also acts directly on the exocytotic machinery in neurons and non-neuronal cells [G. Lonart et al., Neuron 21:1141 (1998); E. Renstrom et al., J. Physiol. 502:105 (1997); K. Yoshimura, Biochim. Biophys. Acta, 1402:171(1998)].
During the search by the yeast two-hybrid screen (i.e., a method for detection of the interaction between two proteins in yeast cells) for an intracellular signaling molecule directly coupling to a sulphonylurea receptor, a component of pancreatic β-cell ATP-sensitive K+ (KATP) channels [N. Inagaki et al. Proc. Natl. Acad. Sci. U.S.A. 91,2679 (1994)], a cAMP sensor protein (called “CAMPS”) was identified and it was found that the protein has two putative cAMP binding domains, a Pleckstrin homology domain (PH domain), and a guanine nucleotide exchange factor (GEF) homology domain.
In the course of this study, two study groups independently reported cAMP binding proteins that activate Rap1, a member of the small G binding proteins [J. de Rooiji et al. Nature 396:474 (1998); H. Kawasaki et al. Science 282:2275 (1998)], and CAMPS was incidentally revealed to be a mouse homologue of cAMP-GEFII [H. Kawasaki et al. Science 282:2275 (1998)].
Though the mechanisms of intracellular vesicle transport system have thus gradually been clarified, substantial part of them remains still unknown. Further progress is needed for the understanding of the mechanisms so as to provide diagnostic agents or therapeutics for a variety of diseases which involve neurons or endocrine cells.
Unlike the former suggestion that only a single cAMP binding domain was present in cAMP-GEFII, the study by the present inventors suggested the presence of two putative cAMP binding domains (cAMP-A and cAMP-B), based on a sequence alignment of cAMP-GEFII sequence and regulatory subunits of PKA. FIG. 1 shows the sequence alignment of the cAMP binding domains. The cAMP binding domains A and B (cAMP-A and cAMP-B, respectively) of cAMP-GEFII and the cAMP binding domains A and B of the PKA regulatory subunit I α (RI α-A and RI α-B, respectively) are shown. The invariant residues in the different cAMP-binding domains are indicated by black boxes.
As shown in FIG. 2, a glutathione-S-transferase (GST)-cAMP-A fusion protein bound to [3H]cAMP with a dissociation constant (Kd) of ˜10 μM, while the binding of [3H]cAMP to a GST-cAMP-B fusion protein was not evident under the same conditions.
FIG. 2 shows the binding of cAMP to cAMP-A. GST-cAMP-A (filled circles) or GST-PKA RI α (open circles) was incubated with different concentrations of [3H]cAMP (0-50 μM). The data for cAMP-A or PKA RI α are normalized relative to maximal cAMP binding activities. Kd values are 10.0±2.3 μM and 23.7±0.6 nM for cAMP and PKA RI α, respectively.
In the cAMP-B domain, the amino acid residue 423, which originally is glutamic acid (Glu), is substituted with lysine (Lys). This glutamic acid residue is important for cAMP binding. Considering that a more rapid dissociation than the wild-type was observed with a PKA regulatory subunit having an equivalent mutation (E-200-K), cAMP-B may also dissociate cAMP rapidly. Thus, a possibility remains that cAMP binds to the cAMP-B domain.